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Cell-Free Protein Synthesis (CFPS) offers many advantages for the production of recombinant therapeutic proteins using the CHO cell-free system. However, many complex proteins are still difficult to express using this method. To investigate the current bottlenecks in cell-free glycoprotein production, we chose erythropoietin (40% glycosylated), an essential endogenous hormone which stimulates the development of red blood cells. Here, we report the production of recombinant erythropoietin (EPO) using CHO cell-free system. Using this method, EPO was expressed and purified with a twofold increase in yield when the cell-free reaction was supplemented with CHO microsomes. The protein was purified to near homogeneity using an ion-metal affinity column. We were able to analyze the expressed and purified products (glycosylated cell-free EPO runs at 25-28 kDa, and unglycosylated protein runs at 20 kDa on an SDS-PAGE), identifying the presence of glycan moieties by PNGase shift assay. The purified protein was predicted to have ∼2,300 IU in vitro activity. Additionally, we tested the presence and absence of sugars on the cell-free EPO using a lectin-based assay system. The results obtained in this study indicate that microsomes augmented in vitro production of the glycoprotein is useful for the rapid production of single doses of a therapeutic glycoprotein drug and to rapidly screen glycoprotein constructs in the development of these types of drugs. CFPS is useful for implementing a lectin-based method for rapid screening and detection of glycan moieties, which is a critical quality attribute in the industrial production of therapeutic glycoproteins.

The kinetic and regulatory properties of enzymes involved in the biosynthesis of UDP-D-galactose, UDP-N-acetylglucosamine, GDP-a-D-mannose and GDP-P-L-fucose from D-glucose 6-phosphate in various reticulo-endothelial tissues was studied. The tissues examined include bovine liver, thyroid, spleen, salivary gland, lung, intestine and mesenteric, pulmonary, portal and sub-maxillary lymph nodes. The maximum rates of specific enzymes in these pathways which were slow enough to be rate-limiting in the formation of glycoproteins in these tissues was determined. UDP-D-galactose 4-epimerase was consistently the rate-limiting reaction in the conversion of D-glucose 6-phosphate to UDP-D-galactose in all of the tissues examined. The series of reactions leading to the formation of GDP-a-D-mannose and GDP-P-L-fucose were limited by the activity of GDP-a-D-mannose pyrophosphorylase and GDP-a-D-mannose oxidoreductase, respectively. The formation of UDP-N-acetylglucosamine was limited by the rate of the amination reaction which converts D-fructose 6-phosphate to D-glucosamine 6-phosphate in the presence of glutamine. Several of these rate-limiting enzymes were partially purified from mesenteric lymph node extracts, and their regulatory properties were examined. GDP-a-D-mannose was found to be a competitive inhibitor of GDP-a-D-mannose pyrophosphorylase. The apparent K, for G T P was 0.06 mM and the Ki for GDP-a-D-mannose was 0.03 mM. The concentrations of GTP and GDP-a-D-mannose in lymph node extracts were determined to be 0.095 and 0.012 pmol per g, respectively. UDP-N-acetylglucosamine and UDP-D-glucose inhibited D-fructose 6-phosphate amidotransferase in a manner competitive with D-fructose 6-phosphate. The K, for fructose 6-phosphate was 0.3 mM, while the Ki for UDP-D-glucose and UDP-Nacetylglucosamine were determined to be 0.4 mM and 0.045 mM, respectively. The concentrations of these metabolites in lymph node tissue were; UDP-D-glucose, 0.42; UDP-N-acetylglucosamine 0.095; and D-fructose 6-phosphate, 0.073 pmol per g wet weight of tissue.The results obtained in these studies show that specific rate-limiting enzymes in the pathways for the biosynthesis of nucleoside diphosphate sugars in reticulo-endothelial tissues may be subject to cumulative feedback inhibition by the nucleoside diphosphate sugars which are the final products of these systems and the initial precursors of the oligosaccharide units of glycoproteins in these tissues.

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